Activation and expansion of hapten- and protein-specific T helper cells from nonsensitized mice.

Hapten- and protein-antigen-specific T helper cells are usually expanded in vitro from lymphocytes obtained from sensitized animals. In this paper we report on the primary activation and proliferation in vitro of T helper cells from nonsensitized animals by using syngeneic cultured epidermal Langerhans cells as a source of potent antigen-presenting cells. The primary in vitro proliferation was blocked with monoclonal antibodies to Ia molecules, to lymphocyte function-associated antigen 1 (LFA-1), and to L3T4. T helper cell populations sensitized in vitro to haptens and protein antigens showed hapten- and antigen-specific proliferation when restimulated in vitro with spleen cells. Besides its experimental usefulness, in vitro generation of syngeneic specific T helper cells may afford possibilities for adoptive immunotherapy.     

Immune response potential to poly(Tyr,Glu)-poly(DLAla)--poly(Lys) of human T cells of different donors.

Human peripheral blood T cells of normal donors were activated in vitro with autologous adherent cells pulsed with poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [abbreviated (T,G)-A--L]. The "educated" T cells were tested: (i) for their ability to produce a (T,G)-A--L-specific T cell-replacing factor in the cooperation with B cells for antibody responses in vivo or in vitro and (ii) for their ability to proliferate in the presence of a second stimulus of (T,G)-A--L. Results of screening of 66 donors demonstrated that educated T cells of about 50% of the donors produced an active (T,G)-A--L-specific factor, whereas activated cells of only half of the factor producers were capable of proliferating in the presence of the antigen. Thus, as reported for all other species studied, human individuals differ in their response potential to (T,G)-A--L.     

Two separate functions of class II (Ia) molecules: T-cell stimulation and B-cell excitation.

We have evaluated the role of major histocompatibility complex-encoded class II (Ia) molecules as transmembrane signaling receptors in the T helper cell-dependent activation of B lymphocytes. For these studies, we utilized the murine B-cell lymphoma CH12, which expresses both I-A and I-E class II molecules. In addition, CH12 cells carry IgM of known antigen specificity and require both specific antigen and Ia-restricted T-cell help for the induction of antibody secretion. In this respect, they resemble normal resting B cells. We have studied the ability of antigen-specific or alloreactive T helper cells reactive with either the I-A or the I-E molecules on CH12 to be activated and their ability to stimulate antibody production by CH12. The results show that, although CH12 cells present antigen to T helper cells that interact with either the I-A or the I-E molecules, CH12 cells are stimulated to secrete antibody only by T helper cells reactive with their I-E molecules. Our data demonstrate that class II molecules are transducers of signals for B-cell excitation in addition to serving a restricting function for helper T-cell stimulation. Moreover, the data demonstrate that these two functions, T-cell stimulation and B-cell excitation, are discrete and need not be expressed by the same Ia molecule.     

Cellular analysis of specificity of antibodies and of delayed type hypersensitivity responses toward some structurally related synthetic antigens: Boosting is determined by specificity of T cells

The crossreactivity between the random synthetic polypeptide antigen poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] and its ordered-sequence analogs (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys) [(T-T-G-G)-A--L] and (Tyr-Glu-Tyr-Glu)-poly(DLAla)--poly(Lys) [(T-G-T-G)-A--L] at the level of humoral and cellular responses was studied. For delayed type hypersensitivity responses, (T,G)-A--L-activated T cells could be challenged with the homologous antigen as well as with the ordered analogs. T cells activated by (T-T-G-G)-A--L could be challenged with either the homologous antigen or with (T,G)-A--L but not with (T-G-T-G)-A--L. Similarly, no cross stimulation was observed between (T-G-T-G)-A--L-activated cells and (T-T-G-G)-A--L, whereas (T,G)-A--L could challenge the latter cells to mediate significant responses. Similar but not identical cross reactions were observed when primed spleen cells or lymph nodes were transferred to irradiated recipients that were boosted for the production of antibodies. In contrast to observations at the level of cellular responses, (T-G-T-G)-A--L-primed spleen or lymph node cells could not be boosted with (T,G)-A--L for the production of detectable amounts of antibodies, although boosting with the homologous antigen resulted in significant levels of (T-G-T-G)-A--L-specific antibodies. Transfer experiments in which mixtures of T and B cells, each primed to a different ordered polypeptide antigen, were injected into irradiated recipients showed that successful cooperation occurs provided that the boost is given with the T-cell-specific antigen. The antibodies produced were specific to the antigen used for B-cell priming. The T-cell-B-cell collaboration probably occurs through specific determinants that are shared between the two antigens in which the ordered peptides are attached to the same multichain polymer and that are recognized by both the T and the B cells.     

Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II- restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T- cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM- derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.     

Mucosal T cells provide helper function but do not proliferate when stimulated by specific antigen in lymphogranuloma venereum proctitis in nonhuman primates

To study antigen-specific immune responses of gut-associated T lymphocytes after gastrointestinal infection, Cynomolgus monkeys were inoculated rectally with Chlamydia trachomatis of the L2 [lymphogranuloma venereum (LGV)] strain. Infected monkeys developed a chronic proctitis with the appearance of LGV-specific immunoglobulin G-antibodies in the serum. Lymphocytes isolated from the peripheral blood, the spleen, and draining lymph nodes had a vigorous antigen-specific proliferative response to LGV in vitro. Both T and B cells proliferated in response to stimulation with LGV, but B-cell proliferation was T-cell-dependent, as shown by cell separation techniques and cell-cycle analysis with dual-laser flow cytometry. Lymphocytes isolated from both involved and uninvolved lamina propria did not proliferate in response to LGV stimulation, whereas mitogen-induced proliferation was not different in lamina propria lymphocytes and the other lymphocyte populations. This lack of antigen-specific proliferation was not caused by a suppressor effect of mucosal T cells or monocytes or the absence of antigen-presenting cells. In contrast, lamina propria T lymphocytes from infected animals were able to provide antigen-specific help for polyclonal immunoglobulin synthesis by immune B lymphocytes after stimulation with LGV. Thus, in LGV proctitis in monkeys, mucosal antigen-reactive T cells differ from lymphocytes in other sites in that they can provide helper function, but are not able to proliferate in response to LGV antigens.     

Inhibition of normal B-cell function by human immunodeficiency virus envelope glycoprotein, gp120.

Despite the occurrence of hypergammaglobulinemia in human immunodeficiency virus (HIV) infection, specific antibody production and in vitro B-cell differentiation responses are frequently impaired. In this study, we have examined the effects of HIV envelope glycoprotein gp120 on T-helper cell function for B cells. In the culture system used, B-cell functional responses were dependent on T-B-cell contact, since separation of T and B cells in double chambers by Transwell membranes rendered the B cells unresponsive in assays of antigen-induced B-cell proliferation and differentiation. Cytokines secreted by T cells were also essential, since anti-CD3 monoclonal antibody (mAb)-activated, paraformaldehyde-fixed T-cell clones failed to induce B-cell proliferation and differentiation. Pretreatment of the CD4+ antigen-specific T cells with gp120 was found to impair their ability to help autologous B cells, as determined by B-cell proliferation, polyclonal IgG secretion, and antigen-specific IgG secretion. The gp120-induced inhibition was specific in that it was blocked by soluble CD4. Furthermore, only fractionated small B cells (which are T-cell-dependent in their function) manifested impaired responses when cultured with gp120-treated T cells. Antigen-induced interleukin (IL)-2 and IL-4, but not IL-6, secretion were markedly reduced in gp120-treated T-cell clones. Addition of exogenous cytokines failed to compensate for defective helper function of gp120-treated T cells. The findings in this study indicate that gp120 impairs helper functions of CD4+ T cells by interfering with T-B-cell contact-dependent interaction; the inhibitory effects of soluble envelope proteins of HIV may contribute to the immunopathogenesis of the HIV-associated disease manifestations.     

Antigen presentation by Ia+ B cell hybridomas to H-2-restricted T cell hybridomas.

The Ia+, H-2d BALB/c lymphoma cell line L10.A 2J was fused to T cell-depleted spleen cells from mouse strains bearing other H-2 haplotypes. A portion of the selected hybrids expressed Ia antigens of the normal spleen cell partner in the fusion as evidenced by their presentation of various antigens to a set of antigen-specific I-region-restricted T cell hybridomas. Three cloned hybrids were studied in detail. Antigen presentation was shown to be inhibited specifically by monoclonal anti-Ia antibodies. Both I-A and I-E molecules were expressed, including in the one case examined hybrid I-A and I-E molecules between the H-2d and H-2b haplotypes. These Ia+ B cell hybridomas provide a useful set of tools for studying the role of I-region-encoded molecules in antigen presentation to T cells.     

Genetic control of immune response to a purified Schistosoma mansoni antigen. II. Establishment and characterization of specific I–A and I–E restricted T–cell clones

T-cell lines and clones specific for a partially protective schistosome antigen (9B antigen) were established from mice immunized with such antigen. The H-2 congenic strains B10.A which express both I-A and I-E class II gene products of the major histocompatibility complex (MHC) and B10.A(4R) which only express I-A molecules were used in these studies. The specific T-cell lines recognized the 9B antigen in the context of either A or E molecules, but both class II antigens were necessary for maximal stimulation of the T-cell lines in lymphocyte proliferation assays. T-cell clones were derived from these lines and their MHC restriction was investigated. Both I-A and I-E restricted clones could be isolated. All clones were specific for 9B antigen showing different degrees of cross-reactivity with a total schistosome extract (CA sonicate). A correlation between the fine specificity of the clones and the expression of class II antigens was demonstrated. Clones specific for 9B antigen, or which reacted to the same extent with 9B antigen and CA sonicate, were I-A restricted, whereas clones which proliferated more in the presence of CA sonicate were all I-E restricted. This suggests that I-E restricted clones recognize more cross-reactive epitopes than I-A restricted clones. These antigen-specific T-cell clones should provide a useful tool for examining the role of class II antigens in the modulation of protective immune response during Schistosoma mansoni infection.     

T cells and human autoimmune thyroid disease: emerging data show lack of need to invoke suppressor T cell problems.

Human T cells recognize self and foreign antigens when such antigens are processed into small peptides and bound to molecules coded for by genes of the HLA region on chromosome 6. The part of the T-cell surface which is responsible for such recognition is a set of molecules coded for by a variety of genes and known as the T-cell-receptor complex. In animal models, T cells are able to transfer autoimmune thyroiditis and T cells have, therefore, long been implicated in the etiology of human autoimmune thyroid disease (AITD). Information gained from the study of intrathyroidal T cells and thyroid antigen-specific T-cell clones has shown that in patients with Graves' disease, mainly helper T-cell clones have been obtained, whereas in autoimmune (Hashimoto's) thyroiditis cytolytic T-cell clones may be predominant. Such thyroid antigen-specific T cells have now been shown to recognize one or other of the three major thyroid-specific antigens; thyroglobulin, thyroid peroxidase, or the TSH receptor and efforts are currently in progress to characterize the T-cell epitopes of these major thyroid autoantigens. Recent findings of restricted T-cell receptor V gene use amongst intrathyroidal T cells confirm the primary role of T cells in human thyroid autoimmune processes leading to AITD. However, the mechanisms whereby such autoreactive T cells escape deletion and anergy, and how they become activated, remain uncertain. There is compelling evidence that the thyroid cell itself, by expressing HLA molecules, and presenting antigen directly to the T cells, may initiate disease, perhaps after an external insult.     

Significance of Lyt phenotypes: Lyt2 antibodies block activities of T cells that recognize class 1 major histocompatibility complex antigens regardless of their function.

The effect of anti-Lyt2 on the generation of helper T-cell function and on cytotoxic effects specific for subregions of the major histocompatibility complex (MHC) was determined. The addition of anti-Lyt2 without complement to in vitro cultures blocked the generation of allogeneic MHC-induced help and lymphokine production and cytotoxic effects when the response was directed against allogeneic class 1 MHC antigens (K and D gene products of the mouse H-2 complex) but had no effect when these responses were specific for class 2 MHC antigens (I region gene products). Anti-Lyt2 failed to block the response of help induced to allogeneic mixed lymphocyte-stimulating determinants or the production of lymphokines by T cells specific for class 1 MHC antigens when concanavalin A lectin was used to induce activity. These and earlier results indicate that the ability of anti-Lyt2 antisera to block function is correlated with T cell specificity for class 1 MHC antigens not with the functional activity of the cells.     

Pathogenic anti-DNA autoantibody-inducing T helper cell lines from patients with active lupus nephritis: isolation of CD4-8- T helper cell lines that express the gamma delta T-cell antigen receptor

The antigen responsible for autoimmunization in systemic lupus erythematosus is unknown. In spite of this obstacle, we show that T helper (Th) cell lines that are functionally relevant to this disease can be established in vitro. We derived a total of 396 interleukin 2-dependent T-cell lines from the in vivo activated T cells of five patients with lupus nephritis. Only 59 (approximately 15%) of these lines had the ability to selectively augment the production of pathogenic anti-DNA autoantibodies that were IgG in class, cationic in charge, specific for native DNA, and clonally restricted in spectrotype. Forty-nine of these autoantibody-inducing Th lines were CD4+ and expressed the alpha beta T-cell receptor (TCR). The other 10 were CD4-8- (double negative), 3 expressing the alpha beta TCR and 7 expressing the gamma delta TCR. All of the autoantibody-inducing Th lines responded to some endogenous antigen presented by autologous B cells. The autoreactive responses of the CD4+ Th lines were restricted to HLA class II antigens, whereas those of the double-negative cells were not. Endogenous heat shock or stress proteins of the HSP60 family that were expressed by the lupus patients' B cells were involved in stimulating an autoreactive proliferation of the gamma delta Th cells. These studies demonstrate a novel helper activity of certain gamma delta T cells in a spontaneous autoimmune response.     

Activated B cells can deliver help for the in vitro generation of antiviral cytotoxic T cells.

The experiments described in this paper show that activated B cells can deliver help for antiviral cytotoxic T (Tc) cell responses in vitro. This conclusion is based on four observations. (i) Influenza viruses induced secondary Tc cell responses in vitro in the absence of CD4+ T cells. This capacity correlated with the B-cell mitogenicity of these viruses. (ii) Depletion of both CD4+ T cells and B cells prevented the generation of anti-influenza Tc cell responses, whereas depletion of either CD4+ T cells or B cells alone failed to do so. In addition, supplementation of unprimed B cells restored the Tc cell responsiveness of primed splenocytes that had been depleted of both CD4+ T cells and B cells. (iii) Contact between T and B cells was not obligatory for the delivery of B-cell helper signal, and hence help was mediated by a soluble factor(s). (iv) Lipopolysaccharide-activated B cells could replace the CD4+ T-cell requirement in the induction of Tc cell responses to nonmitogenic influenza virus in vitro.     

Chronic leukemia with a hybrid surface phenotype (T lymphocytic/myelomonocytic): leukemic cells displaying natural killer activity and antibody-dependent cellular cytotoxicity

A patient with chronic leukemia exhibited uncommon clinical features, such as hypergammaglobulinemia and activation of intravascular coagulation after low-dose irradiation of the enlarged spleen. By light and electron microscopy, the leukemic cells resembled large granular lymphocytes. The following markers were simultaneously expressed on their surface: receptors for sheep erythrocytes and the Fc part of IgG; common T-cell antigens as revealed by a heteroantiserum (HuTLA) and monoclonal antibodies (OKT3, T411); antigens shared by cytotoxic/suppressor T cells (OKT8, T811) as well as myelomonocytic antigens defined by the OKM1 and M522 monoclonal antibodies. The leukemic cells showed both spontaneous (NK) and antibody-dependent (ADCC) cytotoxicity, but they did not suppress B-cell differentiation in vitro.     

Research note: HLA degenerate T-cell epitopes from Plasmodium falciparum liver stage-specific antigen 1 (LSA-1) are highly conserved in isolates from geographically distinct areas

Considerable effort is directed at the development of a malaria vaccine that elicits antigen-specific T-cell responses against pre-erythrocytic antigens of Plasmodium falciparum. Genetic restriction of host T-cell responses and polymorphism of target epitopes on parasite antigens pose obstacles to the development of such a vaccine. Liver stage-specific antigen-1 (LSA-1) is a prime candidate vaccine antigen and five T-cell epitopes that are degenerately restricted by HLA molecules common in most populations have been identified on LSA-1. To define the extent of polymorphism within these T-cell epitopes, the N-terminal non-repetitive region of the LSA-1 gene from Malaysian P. falciparum field isolates was sequenced and compared with data of isolates from Brazil, Kenya and Papua New Guinea. Three of the T-cell epitopes were completely conserved while the remaining two were highly conserved in the isolates examined. Our findings underscore the potential of including these HLA-degenerate T-cell epitopes of LSA-1 in a subunit vaccine.     

Crosstalk between alpha/beta T cells and gamma/delta T cells in vivo: activation of alpha/beta T-cell responses after gamma/delta T-cell modulation with the monoclonal antibody GL3.

Although gamma/delta T cells express numerous in vitro functions similar to alpha/beta T cells, little is known about their biological functioning in vivo. Furthermore, it is unclear whether alpha/beta T cells and gamma/delta T cells act independently or in a coordinated way. In the present study, gamma/delta T cells were modulated in vivo by i.p. injection of the anti-gamma/delta T-cell receptor (TCR) monoclonal antibody GL3. GL3 administration caused disappearance of the gamma/delta TCR in spleen and lymph node cells and the gamma/delta TCR was reexpressed after in vitro cultivation for a few days. When cultured in vitro for 4 days, in the absence of foreign antigens, spleen and lymph node alpha/beta T cells from GL3-modulated mice showed vigorous proliferative responses. CD4 T lymphocytes from GL3-modulated mice produced interleukin 2, and CD8 T cells developed into cytolytic T lymphocytes in vitro capable of lysing syngeneic and allogeneic targets. Treatment with heat-inactivated GL3 or with normal hamster immunoglobulin did not cause any of these effects. These findings suggest that the anti-gamma/delta TCR monoclonal antibody GL3 modulates gamma/delta T cells in vivo and that this modulation has profound effects on alpha/beta T-cell reactivity. Hence, the data suggest a role for gamma/delta T cells in the regulation of alpha/beta T-cell activation in vivo.     

CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.     

Human T cell clones reactive with hepatitis B virus (HBV) surface antigen from a HBV vaccine recipient

Five T-cell clones reactive with HBsAg were obtained from peripheral blood lymphocytes of a HBV vaccine recipient by means of in vitro stimulation of lymphocytes for 5 days by HBsAg and subsequent re-culture of lymphocytes with HBsAg, feeder cells and T cell growth factor (TCGF) for 6 days, followed by the limiting dilution method. Clones, and subclones derived from one original clone showed a specific proliferative response to HBsAg in the presence of autologous feeder cells but not to unrelated antigens, such as influenza virus antigens, herpes simplex antigens, toxoplasma antigens, and PPD. All clones were found to have a surface marker of helper/inducer phenotype, Leu 1+, Leu 2a-, and Leu 3a+, detected by an indirect immunofluorescence method. These clones, however, did not show a substantial helper effect for in vitro anti-HBs production by autologous B cells. This suggests that the mechanism of the helper effect by specific T-cell clones is implicated, and functions of such clones other than specific helper effect on antibody-producing B cells must be considered.     

Suppressor T cells in the specific control of the carrier response to schistosomula in mice infected with Schistosoma mansoni

The carrier effect, using TNP-labelled schistosomula was used to measure the helper T-cell activity against the schistosomula surface in CBA mice exposed to 30 cercariae of Schistosoma mansoni. After infection the helper T-cell activity reached a peak in 8--10 days, but by 6 weeks it had declined to background levels. Five x 10(7) spleen cells from chronically (12-week) infected mice when injected into 9-day infected mice caused a specific suppression of the helper T-cell response to schistosomula. Subsequent fractionation of the spleen cell population using a nylon wool column and specific depletion of T cells from the spleen cell population with anti-Thy 1.2 antisera and complement, showed that the suppressive activity was due to T cells. We conclude that during infection of mice with S. mansoni a population of suppressor T cells is generated which partially regulates antibody production against schistosome surface antigens.     

Selective adsorption of human CD4 T cells

The pathogenesis of most autoimmune diseases directly involves CD4(+) helper T cells. To remove CD4(+) T cells selectively from the circulation, we designed a new column in which an anti-CD4 monoclonal antibody was immobilized on the activated substance. Nearly 90% of CD4(+) T cells were selectively adsorbed from whole blood with a single passage through the column in vitro, resulting in depletion of the antigen-specific T cell responses. We conclude that this new column would be potentially useful for treatment of T cell-mediated autoimmune diseases.